1
Marginal Zone B Cell Depletion Impairs Murine Host Defense Against Borrelia 1
burgdorferi Infection 2
Running Title: Marginal zone B cells in murine Lyme borreliosis 3
Alexia A. Belperron, Department of Internal Medicine, Yale University School of 4
Medicine, New Haven, CT 06520 5
Catherine M. Dailey, Department of Internal Medicine, Yale University School of 6
Medicine, New Haven, CT 06520 7
Carmen J. Booth, Section of Comparative Medicine, Yale University School of 8
Medicine, New Haven, CT 06520 9
Linda K. Bockenstedt*, Department of Internal Medicine, Yale University School of 10
Medicine, New Haven, CT 06520
Section of Rheumatology, Department of Internal Medicine 11
Yale University School of Medicine 12
P.O. Box 208031, New Haven, CT 06520 13
Email address: [email protected] 14
Tel: 203-785-2453 15
Fax: 203-785-7053 16
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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00422-07 IAI Accepts, published online ahead of print on 30 April 2007
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Abstract 17
Marginal zone B (MZB) cells are a B cell subset that produces T cell independent 18
antibodies to bloodborne antigens. In this study, we examined the effects of MZB cell 19
depletion on the immune response to the Lyme disease spirochete Borrelia burgdorferi, 20
an extracellular pathogen for which T cell independent antibody is an important host 21
defense. MZB cell depletion of C3H/HeJ mice using mAb to LFA-1 and alpha4beta1 22
integrins reduced B. burgdorferi-specific IgM titers, enhanced pathogen burden and led 23
to more severe arthritis assessed within the first two weeks of infection. In addition, 24
MZB cell-depleted mice had reduced levels of B. burgdorferi-specific IgG, which 25
correlated with diminished splenic CD4+ T cell activation, proliferation and cytokine 26
production. Passive transfer of immune mouse serum from infected control mice into 27
infected MZB cell-depleted mice reduced pathogen burden, but did not alter the 28
expression of T cell activation markers on splenic CD4+ T cells. These findings 29
demonstrate that MZB cells are not only a source of pathogen-specific IgM important for 30
limiting spirochete burden and pathology, but also play a prominent role in the priming of 31
splenic T cell responses to a bloodborne pathogen.32 ACCEPTED
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Introduction 33
Spirochetes of the genus Borrelia are arthropod-borne agents of both human and 34
domestic animal diseases and are transmitted to vertebrate hosts during vector feeding (4). 35
Borrelia burgdorferi is the etiologic agent of Lyme disease, the most common vector-borne 36
disease in the United States (25). After initial deposition in the skin during tick feeding, B. 37
burgdorferi replicate locally and then disseminate through the blood and tissues to distant 38
sites where disease can be seen. The transient bloodborne phase of B. burgdorferi infection 39
within the vertebrate host promotes wider colonization of the skin, thereby facilitating 40
acquisition of the pathogen by feeding ticks (21). Bloodborne infection, however, can lead to 41
systemic manifestations of disease, including arthritis, myocarditis and neurologic disease 42
(25). Control of the pathogen during the early hematogenous stage of infection is an 43
important host defense against the development of more widespread pathologic sequelae 44
(27). 45
Laboratory mice experimentally infected with B. burgdorferi by intradermal inoculation 46
develop systemic infection, arthritis and myocarditis and serve as a model for acute 47
disseminated Lyme disease in humans (5). Within 4 days of intradermal inoculation, 48
spirochetes become bloodborne and induce myocarditis and inflammatory arthritis that peak 49
in severity by 14-30 days of infection (5). Spirochetes are most abundant in tissues during 50
periods of disease, and can be detected in the bloodstream by culture throughout this period. 51
Both myocarditis and arthritis undergo immune-mediated regression by 45-60 days of 52
infection even though spirochetes persist at low levels in multiple tissues, including hearts 53
and joints. Humoral immunity is an essential mammalian defense against Borrelia 54
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spirochetes (1, 6) and is required for resolution of B. burgdorferi-induced arthritis (6). 55
Antibodies produced in the absence of T cell help are sufficient to prevent infection and to 56
induce arthritis resolution, as has been shown in passive immunization of SCID mice and in 57
experimental infection of mice deficient in T cells, MHC Class II molecules or CD40, a 58
molecule required for T cell-dependent B cell responses (13, 20). The sources of protective 59
and disease resolving antibodies have not been fully delineated, but likely involve innate and 60
follicular B cell subsets to varying degrees throughout the course of infection (6, 20). 61
Marginal zone B (MZB) cells comprise an innate B cell subset localized in mice to the 62
marginal sinuses of the spleen, where they provide early immune responses to bloodborne 63
particulate Ag (19, 22). These cells serve to bridge the innate and adaptive immune systems 64
because they have the capacity to respond to specific foreign Ag more rapidly than follicular 65
B cells (26). In addition to being an early source of pathogen-specific T cell-independent 66
IgM, MZB cells are potent APC that can present particulate Ag to activate naïve CD4+ T 67
cells after immunization (3, 17, 24). MZB cells can be distinguished from follicular B cells 68
by their high levels of CD1d and the complement receptor CD21 expression (2) and lower 69
levels of CD23, a negative regulator of B cells. Reduced levels of CD23 on MZB cells may 70
allow them to produce a more rapid Ab response in comparison to follicular B cells, as has 71
been reported with inert particles and heat-killed bacteria as Ag (22). 72
Using the relapsing fever spirochete, B. hermsii, we have previously shown that MZB 73
cells directly associate with bloodborne Borrelia spirochetes and are activated in vivo to 74
produce pathogen-specific IgM within 72h of infection (7). In the absence of CD1d, 75
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however, MZB production of B. hermsii-specific IgM was impaired, resulting in an 76
accentuated bacteremic phase (7). Paradoxically, MZB cells from CD1d-deficient mice had 77
prolonged upregulation of activation markers, including CD25 and B7.1, in comparison to 78
wildtype MZB cells, yet their production of pathogen-specific IgM in vitro and in vivo was 79
reduced (7). We had previously observed diminished resistance to B. burgdorferi infection 80
and arthritis in CD1d-/- mice, with spirochete DNA found at earlier time points in urinary 81
bladders after intradermal infection in comparison to similarly infected wild-type mice (16). 82
This altered containment of the pathogen during the first week of infection in CD1d-/- mice 83
may be the result of impaired production of B. burgdorferi-specific IgM by innate B cell 84
subsets, including CD1dhigh MZB cells. 85
In this study, we depleted MZB cells from mice to determine their contribution to 86
host defense against B. burgdorferi and to investigate their role in the induction of 87
adaptive immune responses during the first 2 weeks of B. burgdorferi infection. In 88
addition to reduced B. burgdorferi-specific IgM, elevated pathogen burden and earlier 89
appearance of arthritis, we found reduced levels of B. burgdorferi-specific IgG that 90
correlated with diminished splenic CD4+ T cell responses. These results demonstrate a 91
role for MZB cells not only in Borrelia-specific IgM production but also in promoting the 92
early adaptive T and B cell responses to B. burgdorferi infection. 93
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Materials and Methods 94
Mice 95
C3H/HeJ mice, 6-8 weeks in age, were obtained from The Jackson Laboratory (Bar 96
Harbor, ME). Mice were housed in filter frame cages and administered food and water ad 97
libitum according to Yale University animal care and use guidelines. Mice were euthanized 98
by carbon dioxide asphyxiation. The Yale University Institutional Animal Care and Use 99
Committee approved all procedures. 100
Infection of mice 101
Frozen aliquots of low passage B. burgdorferi N40 were grown to logarithmic phase in 102
BSK-H media (Sigma-Aldrich, St. Louis, MO) and enumerated by darkfield microscopy 103
using a Petroff Hausser chamber. Each mouse was inoculated intradermally with 104 104
spirochetes in 100 µl of BSK-H. At the time of mouse sacrifice, infection was confirmed by 105
culturing the blood or a portion of the urinary bladder in BSK-H media for 14 days, after 106
which the presence of spirochetes was determined by darkfield microscopy. 107
Flow cytometry analysis of MZB cells and T cells 108
Splenocytes were isolated from mice at the time of sacrifice. After red blood cell 109
lysis and Fc receptor blockade with anti-CD16/32 mAb (BD Pharmingen, San Diego, 110
CA), MZB cells were identified by flow cytometry using 4-color immunofluorescence 111
staining. Splenocytes were labeled with allophycocyanin-conjugated anti-B220, PE-112
conjugated anti-CD23, and FITC-labelled anti-CD21/CD35 mAb, which allow MZB 113
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cells to be distinguished from follicular B cells (BD Pharmingen). The MZB cell 114
population was identified by its B220+, CD23 low/negative, CD21high phenotype, and the 115
follicular B cell population by its B220+, CD23 high, CD21intermediate phenotype using a 116
FACS Calibur flow cytometer (BD Pharmingen). T cells were labeled with 117
allophycocyanin or cychrome-conjugated anti-Thy1.2 and PE-conjugated or cychrome-118
conjugated anti-CD4 mAb. The T cell activation marker CD44 was identified with either 119
cychrome-conjugated anti-CD44 or biotinylated anti-CD44 mAb detected with 120
cychrome-conjugated streptavidin (BD Pharmingen). The CD45Rb activation marker 121
was detected with FITC-CD45Rb, and the CD62L activation marker was detected with 122
allophycocyanin-conjugated anti-CD62L mAb. All conjugated mAb and streptavidin 123
were used at a 1:50 dilution. 124
MZB Cell Depletion 125
MZB cells were depleted from mice with a single treatment of 100 µg each of anti-126
LFA (!L"2) and anti-CD49d (anti-!4) (BD Pharmingen) as described (7, 18). Controls 127
were treated with isotype-matched mAb (Pharmingen). In each experiment, the efficacy of 128
depletion was assessed by flow cytometry of splenocytes from one mouse sacrificed 7 days 129
after Ab treatment (at the start of infection), as described above. 130
ELISA 131
B. burgdorferi-specific ELISA were performed using sera from infected animals. 132
Ninety-six-well microtiter plates were coated with B. burgdorferi lysate (3µg in 50 µl 100% 133
ethanol per well) by overnight incubation at 4oC. After blocking in PBS containing 5% 134
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BSA, serial two-fold dilutions of sera in PBS, 0.5% BSA, 0.5% Tween 20 were added to the 135
wells and incubated for 1h at room temperature. Secondary biotinylated anti-mouse IgM 136
and IgG (Vector Labs, Burlingame, CA) were used at a 1:1000 dilution, and bound Ab were 137
detected with the ABC Elite peroxidase detection and ABTS substrate kits (Vector Labs). 138
The READY-SET-GO! Mouse Interferon Gamma ELISA kit (eBioscience, San Diego, CA), 139
was used according to the manufacturer’s instructions to measure IFN# in culture 140
supernatants. The concentration of IFN# was determined in serial two-fold dilutions using 141
a standard curve, and the average values were determined for each sample. 142
Quantitative PCR of B. burgdorferi DNA 143
DNA was isolated from 100 µl of whole blood using the Isoquick DNA isolation kit 144
(ORCA Research Inc., Bothwell, WA), and from urinary bladder using the DNeasy kit 145
(Qiagen Inc, Valencia, CA) according to the manufacturer’s instructions. The copy number of 146
B. burgdorferi in each sample was determined by quantitative PCR of the B. burgdorferi recA 147
gene using an iCycler (Biorad, Hercules, CA), the Brilliant SYBR Green kit (Stratagene, 148
Cedar Creek, TX), and the following primers : 5’ primer 149
5’GTGGATCTATTGTATTAGATGAGGCTCTCG3’, 3’ primer 150
5’GCCAAAGTTCTGCAACATTAACACCTAAAG3’. The recA copy number was 151
normalized to the mouse actin gene amplified using the following primers and probe: 5’ 152
primer 5’ATCAGGTAGTCGGTCAGG3’; 3’ primer 5’GGTATCTATCTCGACTC3’, probe 153
6FAM-TCCAGCAGATCTGGATCAGCAAGCATA-MRA (Applied Biosystems, Foster 154
City, CA). A 60o C annealing temperature and forty-five or fifty cycles were used for the recA 155
and actin reactions, respectively. Standard curves were generated for both PCR reactions 156
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using known quantities of DNA. Reactions were performed in duplicate and the quantities of 157
PCR products generated were determined from the standard curves. 158
Histopathology 159
Bilateral hindlimb joints (knee and tibiotarsal joints) were processed and stained with 160
hematoxylin and eosin by routine histologic techniques performed by HSRL Laboratories 161
(Mount Jackson, VA) or the Section of Comparative Medicine, Yale School of Medicine. The 162
knee and tibiotarsal joints were scored for arthritis severity on a scale of 0 (negative) to 3 163
(severe) in a blinded fashion as described (6). 164
Passive immunization 165
Groups of 4 or 5 control and MZB cell-depleted mice were inoculated intradermally 166
with 104 N40 B. burgdorferi spirochetes. At 10d of infection, groups of WT and MZB cell-167
depleted mice were inoculated intraperitoneally with either 200 µl of serum from the 10d-168
infected control mice or NMS, diluted with PBS to a final volume of 300 µl. A third group 169
received PBS alone. Four days after passive immunization, mice were sacrificed and T cell 170
activation, spirochete burdens and Ab titers were measured as described above. 171
Splenocyte culture and ex vivo T cell activation 172
Spleens were fragmented between the frosted ends of two glass slides to retrieve 173
splenocytes. After red blood cell lysis, the splenocytes from mice within the isotype control 174
group were combined, and the splenocytes from each MZB cell-depleted mouse were 175
labeled according to the manufacturer’s instructions with carboxy-fluorescein diacetate, 176
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succinimidyl ester (CFSE) using the Cell Trace CFSE Proliferation Kit (Molecular Probes, 177
Eugene Oregon). The labeled cells from the combined control group and each MZB cell-178
depleted mouse were aliquoted into 24-well tissue culture plates at a density of 5x106 cells 179
in 1 ml of Click's media (Irvine Scientific, Santa Ana, CA) supplemented with 10% FBS, 180
penicillin (100 U/ml) and streptomycin (100 pg/ml). Cells were stimulated for 72h with 10 181
µg/ml of B. burgdorferi sonicate or PBS, after which they were stained with 182
immunofluorescent Ab for analysis by flow cytometry as described above. The cell 183
supernatants were collected and stored at -80 o C for later cytokine analysis by ELISA, as 184
described above. 185
Results 186
Depletion of MZB cells decreases B. burgdorferi-specific Ab titers and enhances 187
arthritis early in infection. To examine the role of MZB cells in host resistance to B. 188
burgdorferi in its bloodborne phase, we treated mice with mAb to LFA-1 and alpha4beta1 189
integrins, two integrins that tether MZB cells to the marginal sinuses in the spleen. This 190
treatment results in selective depletion of MZB cells without affecting other lymphocyte 191
populations in the spleen (7, 18). The treatment regimen used results in MZB cell 192
depletion for 2-3 weeks, after which time newly generated MZB cells from the bone 193
marrow begin to repopulate the spleen. Mice were infected with B. burgdorferi one week 194
after mAb treatment, a time previously determined to correlate with at least an 80% 195
reduction in the number of MZB cells (18). Mice depleted of MZB cells had lower B. 196
burgdorferi-specific IgM levels in the serum 7d after infection (Figure 1A) and had 197
increased numbers of spirochetes in the blood (Figure 1B), similar to our previous 198
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findings in mice infected with B. hermsii spirochetes (7). B. burgdorferi-specific IgG 199
titers were also reduced in MZB cell-depleted mice in comparison to infected control 200
mice treated with isotype matched mAb (Figure 1C). Enhanced pathogen burden in MZB 201
cell-depleted mice was associated with more severe arthritis assessed at 10 and 14 days of 202
infection (Table I). 203
Depletion of MZB cells impairs T cell activation and pathogen specific IgG 204
production. MZB cells produce pathogen-specific Ab within a few days of bloodborne 205
infection, after which time follicular B cells become activated to produce Ab. After day 7 206
of infection, IgM titers began to increase but the IgG titers remained low in MZB cell-207
depleted mice. At day 10 of infection, the serum B. burgdorferi-specific IgM titers were 208
comparable to those of infected control mice (reciprocal endpoint IgM titer, MZB-209
depleted mice = 1067±267, isotype control mice= 800±400), and trended higher at 14 210
days of infection (1360±538 vs 720±180, respectively). The B. burgdorferi-specific IgG 211
levels, however, were still reduced at day 14 (MZB cell-depleted mice = 2720±999 vs 212
isotype control mice = 8320±1920, P = .032). All IgG subtypes were represented (data 213
not shown). Because the serum titers of B. burgdorferi-specific T cell-dependent Ab 214
were reduced in MZB cell-depleted mice, we examined the expression levels of the T cell 215
activation markers CD44, CD45Rb and CD62L (11, 15). When examined directly ex 216
vivo, splenic CD4+ T cells from 7d infected control mice had increased expression of 217
CD44 in comparison to T cells from MZB cell-depleted mice (Figure 2A). The 218
differences in CD44 expression were still apparent at 14 days of infection (Figure 2B), at 219
which time CD45Rb expression was diminished to a greater extent on T cells from 220
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infected control mice when compared to those from infected MZB cell-depleted mice 221
(Figure 2C). No differences were noted in the expression of CD62L, which was 222
diminished on T cells from both infected control and MZB cell-depleted mice (Figure 223
2D). T cells from 7d-infected MZB cell-depleted mice stimulated in vitro with B. 224
burgdorferi lysate did not alter expression of CD44 (Figure 3A) or CD45Rb (Figure 3B) 225
in comparison to unstimulated cells from either infected control or infected MZB cell-226
depleted mice. In contrast, stimulated T cells from infected control mice exhibited an 227
increase in CD44 and decrease in CD45Rb expression. In vitro stimulation also reduced 228
CD62L expression by T cells from infected MZB cell-depleted mice to a lesser degree 229
when compared to T cells from infected control mice (Figure 3C). T cells from 14d-230
infected MZB cell-depleted mice stimulated in vitro with B. burgdorferi lysate still 231
expressed low levels of the activation marker CD44, whereas this marker could be 232
detected on a significant proportion of stimulated T cells from infected control mice 233
(Figure 3D). The levels of CD62L and CD45Rb, however, were similarly diminished on 234
T cells from both groups of mice after in vitro stimulation (data not shown). 235
T cells from B. burgdorferi-infected MZB-cell depleted mice proliferate less and 236
produce lower amounts of IFN! in comparison to T cells from infected control mice. To 237
assess the effects of MZB cell depletion on T cell proliferation, splenocytes from 7d-238
infected mice were labeled with CFSE and stimulated in vitro with B. burgdorferi lysate 239
for 72h. A higher percentage of splenic CD4+ T cells from control mice proliferated in 240
response to B. burgdorferi lysate stimulation and those cells underwent more divisions 241
than cells from MZB cell-depleted mice (Table II). Stimulated splenic CD4+ T cells from 242
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MZB cell-depleted mice proliferated to a similar extent as unstimulated cells from 243
infected mice, with 74.7% and 75.3% of unstimulated cells remaining quiescent from 244
control and MZB cell-depleted mice, respectively. CD4+ T cells from 7, 10, and 14d-245
infected MZB cell-depleted mice stimulated with B. burgdorferi lysate produced lower 246
levels of IFN# in comparison to infected control mice (Table III). IFN# could not be 247
detected in culture supernatants of unstimulated splenocytes from either infected control 248
or MZB cell-depleted mice (data not shown). The percentages of CD4+ T cells in the 249
cultured splenocytes were not statistically different between MZB cell-depleted and 250
control mice (Table III). 251
B. burgdorferi infected MZB cell-depleted mice have elevated pathogen burdens 252
that can be reduced by immune serum from infected control mice. To assess the effects of 253
reduced B. burgdorferi-specific Ab titers on pathogen burden, we measured B. 254
burgdorferi DNA in urinary bladders of mice 14d after infection. Urinary bladders were 255
chosen because they are a representative site of disseminated infection and are 256
reproducibly positive by culture at this stage of infection. At 14d, even though the B. 257
burgdorferi-specific IgM titers were comparable in MZB cell-depleted and control mice, 258
the pathogen burdens remain elevated in the urinary bladders of MZB-depleted mice 259
(Figure 4A). To determine whether the elevated pathogen burden was a consequence of 260
the reduced anti-B. burgdorferi-specific Ab titers, we treated 10d-infected MZB-depleted 261
mice with immune serum from similarly infected WT mice. Four days after treatment, 262
the immune sera did in fact lower the pathogen burden in the MZB-depleted mice to 263
levels found in the control mice, whereas NMS and PBS had no effect (Figure 4B). 264
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Discussion 265
MZB cells are a first-line defense against bloodborne pathogens entering the spleen 266
freely through the blood or transported by circulating APCs. These cells are poised to 267
secrete IgM and IgG3 to foreign antigens before the development of adaptive immune 268
responses. In addition, recent immunization studies have demonstrated that MZB cells 269
can transport immune complexes to follicular dendritic cells and deposit them on their 270
surface (12), or process Ag for direct presentation on MHC Class II molecules to naïve 271
CD4+ T cells (3). Few studies, however, have investigated the response of MZB cells 272
during the course of an active infection. Borrelia spirochetes are a particularly suitable 273
pathogen with which to examine MZB cell involvement in host defense because they 274
have a distinct bloodborne phase in the mouse and T cell independent Ab is sufficient to 275
control infection. Using B. hermsii infection of mice, which manifests as periodic 276
episodes of bacteremia in which spirochetes are visible in the blood, we previously 277
showed that MZB cells produce pathogen-specific IgM and upregulate T cell 278
costimulatory molecules within 72h of infection (7). In the current study, we sought to 279
determine the contribution of MZB cells to the evolving host response to B. burgdorferi, 280
a subacute pathogen in mice that becomes bloodborne within 4 days of intradermal 281
inoculation, but rapidly exits the vasculature to infect other tissues. We used mAb to 282
selectively deplete MZB cells from normal mice prior to infection so as to avoid 283
confounding issues of immunologic defects associated with mutant mouse strains that 284
lack MZB cells (14). The mAb employed to deplete MZB cells are not expected to 285
impact B. burgdorferi interaction with host tissue since this organism does not bind LFA-286
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1 or alpha4beta1 integrins (10). The MZB cell depletion is only transient, as newly 287
formed MZB cells repopulate the spleen by day 14 of infection (data not shown, 18). Our 288
results provide evidence that MZB cells are not only an important source of pathogen 289
specific IgM, but they also play a critical role in stimulating T cell-dependent Ab 290
responses during the initial stages of infection. 291
Deficiency in MZB cells led to reduced pathogen specific IgM in the serum at 7d. 292
The low level of IgM detected at this stage of infection may arise from other B cell 293
subsets, such as bone marrow derived perisinusoidal B cells or B1 B cells, that participate 294
in T cell-independent immune responses to bloodborne antigens (9). At later time points, 295
the B. burgdorferi-specific IgM titers approached those of infected control mice and by 296
day 14 were about two-fold higher. The IgM detected at day 14 may arise from newly 297
developed MZB cells that have repopulated the spleen (18), other innate B cell 298
populations, and/or follicular B cells. In contrast, the IgG titers in MZB cell depleted 299
mice were low at day 7 and remained low at day 14 of infection, a time point at which T 300
cell-dependent pathogen-specific Ab responses are normally readily detectable in B. 301
burgdorferi-infected mice. These findings suggested that MZB cell depletion had 302
affected not only the T cell independent B cell response to B. burgdorferi, but also the 303
activation of CD4+ T cells that facilitate B cell isotype switching. 304
MZB cell-depleted mice had impaired splenic T cell responses when examined 305
directly ex vivo and after in vitro restimulation with B. burgdorferi lysate. Although 306
immunization studies have revealed that MZB cells can be potent APC and can directly 307
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activate T helper cells (3), MZB cells have not previously been shown to have a direct 308
impact on T cell-dependent Ab production during infection. MZB cells are capable of 309
transporting and depositing IgM-containing immune complexes onto follicular dendritic 310
cells (12). Both the decrease in the number of MZB cells after depletion and the early 311
decrease in IgM titers would negatively impact this method of Ag presentation and 312
subsequent T cell activation. The phenotype of MZB-cell depleted mice contrasts with 313
that of CD1d-/- mice, even though in both, Borrelia-specific IgM responses are impaired 314
(7). We have previously shown that MZB cells from B. hermsii-infected CD1d-/- mice 315
increase expression of T cell costimulatory molecules, suggesting that CD1d-/- MZB 316
cells are still capable of priming and activating helper T cells. When we directly 317
compared B. burgdorferi-specific IgM and IgG titers from 7d infected wild type, MZB 318
cell-depleted and CD1d-/- mice, we found that IgG titers were preserved in CD1d-/- mice 319
but not MZB cell-depleted mice (reciprocal endpoint positive titer IgM: WT 1000 ± 200, 320
MZB cell-depleted 450 ± 126, CD1d-/- 425 ± 287; IgG: WT 1920 ± 739, MZB cell-321
depleted 720 ± 403, CD1d-/- 2133 ± 739). Thus, in the case of Borrelia infection, the 322
APC contributions of MZB cells to T helper cell activation appears to be CD1d-323
independent but pathogen-specific IgM secretion by MZB cells is CD1d-dependent. 324
Reduction in Borrelia-specific Ab likely contributes to the 5-10 fold increase in 325
pathogen burden noted in the MZB-depleted mice, as Ab is a key defense mechanism 326
against infection with B. burgdorferi. In support of this interpretation, we found that 327
passive transfer of immune serum to infected MZB cell-depleted mice reduced their 328
pathogen load, consistent with the known effects of pathogen-specific Ab on B. 329
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burgdorferi infection in mice (6, 20). Passive immunization, however, had no impact on 330
the reduced levels of activation marker expression by the T cells in MZB cell depleted 331
mice (data not shown). 332
Our results also show that B. burgdorferi infection of MZB cell-depleted mice 333
results in the earlier development of arthritis. In C3H mice, arthritis peaks in severity at 4 334
weeks of infection, but evidence of arthritis can be seen earlier. In our study, all of the 335
joints examined from the 10d-infected MZB-cell depleted mice exhibited arthritis 336
whereas only a few from the control animals had inflammation at that time point, 337
suggesting that MZB cell depletion altered the kinetics of disease development. Even at 338
day 14, a time point when C3H mice normally have developed arthritis, the MZB cell-339
depleted mice had more pronounced disease. B. burgdorferi-induced arthritis severity is 340
known to be equivalent in T cell-deficient and immunocompetent mice, but is more 341
severe in B cell-deficient mice (8, 20, 23). Our results suggest that a deficiency in the 342
MZB cell subset and its production of pathogen specific IgM can exacerbate arthritis 343
early in infection. 344
In summary, we found reduced pathogen-specific IgG and significant impairment in 345
the activation, proliferation and cytokine production by splenic T cells from MZB cell-346
depleted mice infected with B. burgdorferi. These findings reveal an important role for 347
this B cell subset in priming splenic T cells and the subsequent development of T cell 348
dependent IgG responses during infection, extending similar observations after 349
immunization (3). Our study also supports our previous finding that MZB cells are a 350
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source of pathogen-specific IgM during the early stages of Borrelia infection and shows 351
that a deficiency in this response can exacerbate arthritis. These findings may have 352
relevance to other bloodborne pathogens for which the timely production of Ab and/or T 353
cell-mediated immunity are important for limiting pathogen burden and pathologic 354
sequelae in the host. 355
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Acknowledgements 356
We thank Deborah Beck and Jialing Mao for excellent technical assistance. 357
This work was supported by grants from the NIH (AI50604 and AR47058) and the 358
Arthritis Foundation.359
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References 360
1. Alugupalli, K. R., S. Akira, E. Lien, and J. M. Leong. 2007. MyD88- and 361
Bruton's tyrosine kinase-mediated signals are essential for T cell-independent 362
pathogen-specific IgM responses. J Immunol 178:3740-3749. 363
2. Amano, M., N. Baumgarth, M. D. Dick, L. Brossay, M. Kronenberg, L. A. 364
Herzenberg, and S. Strober. 1998. CD1 expression defines subsets of follicular 365
and marginal zone B cells in the spleen: beta 2-microglobulin-dependent and 366
independent forms. J Immunol 161:1710-1717. 367
3. Attanavanich, K., and J. F. Kearney. 2004. Marginal zone, but not follicular B 368
cells, are potent activators of naive CD4 T cells. J Immunol 172:803-811. 369
4. Barbour, A. G., and S. F. Hayes. 1986. Biology of Borrelia species. Microbiol 370
Rev 50:381-400. 371
5. Barthold, S. W., D. S. Beck, G. M. Hansen, G. A. Terwilliger, and K. D. 372
Moody. 1990. Lyme borreliosis in selected strains and ages of laboratory mice. J 373
Infect Dis 162:133-138. 374
6. Barthold, S. W., S. Feng, L. K. Bockenstedt, E. Fikrig, and K. Feen. 1997. 375
Protective and arthritis-resolving activity in sera of mice infected with Borrelia 376
burgdorferi. Clin Infect Dis 25 Suppl 1:S9-17. 377
7. Belperron, A. A., C. M. Dailey, and L. K. Bockenstedt. 2005. Infection-378
induced marginal zone B cell production of Borrelia hermsii-specific antibody is 379
impaired in the absence of CD1d. J Immunol 174:5681-5686. 380
ACCEPTED
on January 22, 2019 by guesthttp://iai.asm
.org/D
ownloaded from
21
8. Bockenstedt, L. K., I. Kang, C. Chang, D. Persing, A. Hayday, and S. W. 381
Barthold. 2001. CD4+ T helper 1 cells facilitate regression of murine Lyme 382
carditis. Infect Immun 69:5264-5269. 383
9. Cariappa, A., I. B. Mazo, C. Chase, H. N. Shi, H. Liu, Q. Li, H. Rose, H. 384
Leung, B. J. Cherayil, P. Russell, U. von Andrian, and S. Pillai. 2005. 385
Perisinusoidal B cells in the bone marrow participate in T-independent responses 386
to blood-borne microbes. Immunity 23:397-407. 387
10. Coburn, J., J. M. Leong, and J. K. Erban. 1993. Integrin alpha IIb beta 3 388
mediates binding of the Lyme disease agent Borrelia burgdorferi to human 389
platelets. Proc Natl Acad Sci USA 90:7059-7063. 390
11. DeGrendele, H. C., M. Kosfiszer, P. Estess, and M. H. Siegelman. 1997. CD44 391
activation and associated primary adhesion is inducible via T cell receptor 392
stimulation. J Immunol 159:2549-2553. 393
12. Ferguson, A. R., M. E. Youd, and R. B. Corley. 2004. Marginal zone B cells 394
transport and deposit IgM-containing immune complexes onto follicular dendritic 395
cells. Int Immunol 16:1411-1422. 396
13. Fikrig, E., S. W. Barthold, M. Chen, C. H. Chang, and R. A. Flavell. 1997. 397
Protective antibodies develop, and murine Lyme arthritis regresses, in the absence 398
of MHC class II and CD4+ T cells. J Immunol 159:5682-5686. 399
14. Guinamard, R., M. Okigaki, J. Schlessinger, and J. V. Ravetch. 2000. 400
Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the 401
humoral response. Nat Immunol 1:31-36. 402
ACCEPTED
on January 22, 2019 by guesthttp://iai.asm
.org/D
ownloaded from
22
15. Huet, S., H. Groux, B. Caillou, H. Valentin, A. M. Prieur, and A. Bernard. 403
1989. CD44 contributes to T cell activation. J Immunol 143:798-801. 404
16. Kumar, H., A. Belperron, S. W. Barthold, and L. K. Bockenstedt. 2000. 405
Cutting edge: CD1d deficiency impairs murine host defense against the 406
spirochete, Borrelia burgdorferi. J Immunol 165:4797-4801. 407
17. Lopes-Carvalho, T., J. Foote, and J. F. Kearney. 2005. Marginal zone B cells 408
in lymphocyte activation and regulation. Curr Opin Immunol 17:244-250. 409
18. Lu, T. T., and J. G. Cyster. 2002. Integrin-mediated long-term B cell retention 410
in the splenic marginal zone. Science 297:409-412. 411
19. MacLennan, I. C., and Y. J. Liu. 1991. Marginal zone B cells respond both to 412
polysaccharide antigens and protein antigens. Res Immunol 142:346-351. 413
20. McKisic, M. D., and S. W. Barthold. 2000. T-cell-independent responses to 414
Borrelia burgdorferi are critical for protective immunity and resolution of lyme 415
disease. Infect Immun 68:5190-5197. 416
21. Nuttall, P. A. 1998. Displaced tick-parasite interactions at the host interface. 417
Parasitology 116 Suppl:S65-72. 418
22. Oliver, A. M., F. Martin, G. L. Gartland, R. H. Carter, and J. F. Kearney. 419
1997. Marginal zone B cells exhibit unique activation, proliferative and 420
immunoglobulin secretory responses. Eur J Immunol 27:2366-2374. 421
23. Schaible, U. E., S. Gay, C. Museteanu, M. D. Kramer, G. Zimmer, K. 422
Eichmann, U. Museteanu, and M. M. Simon. 1990. Lyme borreliosis in the 423
severe combined immunodeficiency (scid) mouse manifests predominantly in the 424
joints, heart, and liver. Am J Pathol 137:811-820. 425
ACCEPTED
on January 22, 2019 by guesthttp://iai.asm
.org/D
ownloaded from
23
24. Song, H., and J. Cerny. 2003. Functional heterogeneity of marginal zone B cells 426
revealed by their ability to generate both early antibody-forming cells and 427
germinal centers with hypermutation and memory in response to a T-dependent 428
antigen. J Exp Med 198:1923-1935. 429
25. Steere, A. C., J. Coburn, and L. Glickstein. 2004. The emergence of Lyme 430
disease. J Clin Invest 113:1093-1101. 431
26. Vos, Q., A. Lees, Z. Q. Wu, C. M. Snapper, and J. J. Mond. 2000. B-cell 432
activation by T-cell-independent type 2 antigens as an integral part of the humoral 433
immune response to pathogenic microorganisms. Immunol Rev 176:154-170. 434
27. Wormser, G. P., D. McKenna, J. Carlin, R. B. Nadelman, L. F. Cavaliere, D. 435
Holmgren, D. W. Byrne, and J. Nowakowski. 2005. Brief communication: 436
hematogenous dissemination in early Lyme disease. Ann Intern Med 142:751-437
755.438
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Figure Legends 439
Figure 1. MZB cell depletion reduces both B. burgdorferi-specific IgM and IgG titers 440
and increases pathogen burden at 7d of infection. Ab titers are the reciprocal end-point 441
positive titers of B. burgdorferi-specific IgM (Panel A) or IgG (Panel C) in serum 442
obtained from 7d-infected control and MZB cell-depleted mice. In Panel B, the pathogen 443
burden in the blood of each mouse was determined by quantitative PCR as described in 444
Materials & Methods; the copy number of the B. burgdorferi recA gene was normalized 445
to 1 ng of the mouse actin gene product. Results were derived from two separate 446
experiments using 4-5 mice per group. For comparison purposes, the relative pathogen 447
burden was set to 1 for the control mouse group in each experiment (the raw values for 448
the control groups were 41,000 and 1,250 copies of B. burgdorferi recA/ng mouse actin, 449
respectively). The pathogen burden calculated for each mouse is the average of 450
duplicates from two separate assays. The differences between the control and the MZB 451
cell-depleted serum titers for IgM (P=0.03) and IgG (P=0.03) as well as the pathogen 452
burdens (P=0.01) were significant (Mann-Whitney test). 453
Figure 2. T cells from infected MZB cell-depleted mice have reduced activation markers 454
in comparison to controls. Splenocytes from 7d (Panel A) and 14d (Panels B, C) infected 455
mice were labeled and analyzed by flow cytometry as described in Materials & Methods. 456
Gates were set on Thy 1.2+, CD4+ cells. The activation marker expression on the gated 457
cells from one representative mouse per group is shown in the histograms: light gray 458
solid histogram ! cells from infected control mouse; black line open histogram " cells 459
from infected MZB cell-depleted mouse; dark gray solid histogram ! cells from 460
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uninfected control mouse; gray line open histogram " cells from uninfected MZB cell-461
depleted mouse. The averages of the mean fluorescent intensities (MFIs) from the 5 462
infected MZB cell-depleted mice were significantly different from the 5 infected control 463
mice at days 7 and 14 of infection (Panel A, P=0.02; Panel B, P=0.01, Panel C, P=0.004, 464
as determined by the two-tailed Student t test). There were no statistical differences in 465
the MFIs between the uninfected MZB cell-depleted and control mouse groups. 466
Figure 3. CD4+ T cells from MZB cell-depleted mice have reduced responses to B. 467
burgdorferi after in vitro stimulation. Splenocytes from 7d (Panels A-C) and 14d (Panel 468
D) infected mice were stimulated with B. burgdorferi lysate for 72h prior to 469
immunofluorescent staining, as described in Materials & Methods. Gates were set on 470
Thy 1.2+, CD4+ cells. The activation marker expression on the gated cells from one 471
representative mouse per group is shown in the histograms: light gray solid histogram ! 472
stimulated cells from infected control mouse; black line open histogram " stimulated 473
cells from infected MZB-cell depleted mouse; dark gray solid histogram ! unstimulated 474
cells from infected control mouse; gray line open histogram " unstimulated cells from 475
infected MZB cell-depleted mouse. The averages of the MFIs from the stimulated cells 476
from 5 MZB cell-depleted mice were significantly different from those of 5 control mice 477
(Panel A, P=0.02; Panel B, P=0.01, Panel C, P=0.003; and Panel D, P=0.005, as 478
determined by a two-tailed Student t test). 479
Figure 4. Passive transfer of immune serum from 10d-infected control mice into MZB 480
cell-depleted mice reduces pathogen burden. 481
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The relative pathogen burden in urinary bladders was determined as described in Figure 1 482
and performed on specimens from two separate experiments in which the control mice 483
had an average of 29,000 and 112,800 copies of B. burgdorferi recA gene/ng mouse actin 484
gene, respectively. At day 14 (Panel A), MZB cell-depleted mice had a significantly more 485
B. burgdorferi DNA when compared to controls (P=0.025, Mann-Whitney test). In Panel 486
B, 10d-infected MZB cell-depleted mice were treated with either immune mouse serum 487
(IMS) from 10d-infected control mice, NMS or PBS and analyzed for pathogen burden at 488
infection day 14. The results from the NMS and PBS treatment groups were combined 489
because no differences were observed. Comparison of groups was performed using the 490
Mann-Whitney test with the following results: P=0.55 for IMS-treated control versus 491
IMS-treated MZB cell-depleted groups; P=0.003 for the combined PBS/NMS-treated 492
control versus PBS/NMS-treated MZB cell-depleted groups; P=0.019 for the IMS-treated 493
MZB cell-depleted group versus the PBS/NMS-treated MZB cell-depleted groups.494
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Table I. Depletion of MZB cells exacerbates arthritis in B. burgdorferi-infected mice 495
Day of Infection Mouse Group Arthritis Prevalencea Mean Arthritis Severityb 496
10 Isotype Control 3/10 0.4 ± 0.7 497
MZB Cell-Depleted 11/11c 1.6 ± 0.7d 498
14 Isotype Control 12/28 0.5 ± 0.7 499
MZB Cell-Depleted 17/23e 1.1 ± 0.9f 500
aArthritis prevalence was reported as the number of joints with inflammation over 501
the total number of joints examined in each group. 502
bArthritis was scored on a scale of 0 (negative) to 3 (severe) and reported as the 503
average score of the inflamed joints in each mouse group. Three to 7 mice were 504
examined in each group. The data for the arthritis at 14 days is combined from 505
two separate experiments. cMZB cell-depleted versus control P=.001, eP=.046, 506
Fisher's exact test; dP=.001, fP=.016 two tailed Student t test.507
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Table II. Proliferation of CD4+ T cells from 7d-infected mice after in 508
vitro restimulationa 509
Number of Cell Divisions 510
Mouse Group 0 1 2 3 511
MZB cell-depleted 76.2b 21.2c 2.03d 0.42e 512
Isotype control 21.0 50.7 25.3 2.08 513
a5x106 CFSE-labeled splenocytes were cultured with 10 µg of B. 514
burgdorferi lysate for 72h. The percentage of CD4+ T cells in each 515
division, based on intensity of CFSE staining, was calculated using 516
Flowjo software (Tree Star Inc. Ashland, OR). The numbers represent 517
the percentage of CD4+ T cells in each generation. P values were 518
determined by a two-tailed Student t test comparing values for MZB cell-519
depleted mice with isotype control mice (bP=0.002; cP=0.016; dP<0.0001; 520
and eP<0.0001).521 ACCEPTED
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Table III. Splenocytes from MZB cell-depleted mice produce less IFN!a 522
Day of Infection 523
Mouse Group 7 10 14 524
MZB cell-depleted 13.1 ± 2.5b 55.4 ± 5.4c 21.6 ± 7.0d 525
Isotype control 74.2 ± 9.8 250 ± 15.0 106.7 ± 31.7 526
a Values are the mean ng/ml ± standard deviation of IFN# in culture 527
supernatants. 5x106 splenocytes were cultured with 10 µg of B. 528
burgdorferi lysate for 72h (days 7 and 10 of infection) or for 48h (day 14 529
of infection). IFN# levels in the cell supernatants were determined by 530
ELISA. P values were determined by a two-tailed Student t test 531
(bP=<0.0001; cP=<0.0001; dP=.002). The percentages of CD4+ T cells in 532
both groups at each time point were similar (57.3 & 50.7, 71.3 & 52.9, and 533
60.8 & 59.4% for MZB cell-depleted and isotype control groups at days 7, 534
10 and 14, respectively). 535 ACCEPTED
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Control MZB-Depl.
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C
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Figure 2
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Rela
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